4 Levels of Protein Structure Lecture

Protein Structure Proteins: the building blocks of life 4 Levels of Protein Structure The STRUCTURE of proteins has been divided into four categories: 1) primary structure – sequence of amino acids 2) secondary structure – small units of repetitive structure 3) tertiary structure – overall 3D shape 4) quaternary structure – shape of ≥2 chains 4 levels of protein structure primary structure – tertiary structure – secondary structure – 4 levels of protein structure In order to understand these levels of structure, you need to understand the nature of the polymer first. In other words, the linkage or PEPTIDE BOND quaternary structure – The Peptide Bond Peptide Bond H2O Amino-acid RESIDUE H2O Peptide Bond The 4 S’s for the Peptide Bond: Shape Size Solubility Stability : Peptide Bond - Shape Resonance of Peptide Bond Consequences: 1) Double-bond Character a) Length b) Strength c) Planarity Peptide Bond - Shape R’ R H R R’ Steric hindrance cis trans Trans vs. Cis of Peptide Bond Peptide Bonds Assume Trans Configuration Conformation or Configuration? Peptide Bond - Size Bond angles all near 120° Bond angles all near 120° C-C=O peptide Ca-N Tetrahedral angles are 109° Peptide Bond - Size ~7 Å 3.63 Å No free rotation of peptide bonds Therefore, there are only TWO bonds in the whole polymer that are flexible: Ca-N is called the Phi (f) Ca-N Ca-C=O Ca-C=O is called the Psi (y) Peptide Bond - Size One Amino-acid ”residue” Three Amino-acid ”residues” in sequence G M D A F S G G V Extended Conformation of Polypeptide Let’s take this tripeptide with two peptide bonds, get small, and sit on this alpha carbon Peptide Bond C V Size )2 H3 (C C H COO– H Ca-C=O is called the Psi (y) Ca-N is called the Phi (f) G Fully extended all trans peptide: f = -180° and y = +180° (H) Angles increase as you turn the peptide-bond plane clockwise R(H) Angles decrease as you turn the peptide-bond plane counter-clockwise H G N Torsion Angles of Polypeptide Backbone + NH3 Peptide Bond - Size Fully extended -180 Ramachandran Diagram 0 +180 y (+180à0°) y went from +180° to smaller angles on Ramachandran Plot Recall: Angles decrease as you turn the peptide-bond plane counter-clockwise Peptide Bond - Size Fully extended -180 Ramachandran Diagram 0 +180 y (+180à0°) y went from +180° to smaller angles on Ramachandran Plot Recall: Angles decrease as you turn the peptide-bond plane counter-clockwise Peptide Bond - Size Only 25% of allowable angles used a • Glycine has more allowable angles (in yellow) Why? 20 Peptide Bond - Solubility d– : d+ Resonance of Peptide Bond Consequences: 1) Double-bond Character a) Length b) Strength c) Planarity 2) Partial charges on O & N – Polarity of peptide bond a) O is excellent H-bond acceptor b) N is excellent H-bond donor This makes the polymer (called “alpha-carbon backbone”) very water soluble Peptide Bond The 4 S’s for the Peptide Bond (recap): Shape Size Solubility Stability –trans & double-bond, planar character due to resonance –7 Å repeat length & two rotatable bonds at Ca (f & y) –highly polar bond due to resonance –high bond energy due to resonance Peptide Bond - Stability What is the energetics of hydrolysis? The DG of hydrolysis is –2.4 kcal/mole (~ –10 kJ/mole) H2O H2O Glycylalanine + H2O ⇌ Glycine + Alanine Why doesn’t your hair fall apart into AA & water, or all other proteins? Kinetically stable, thermodynamically unstable What does it take to hydrolyze proteins? Peptide Bond The catalysis of the hydrolysis reaction to cleave the Peptide Bond: Acid – 6 N HCl, 16 hr at 110 C° All peptide (and amide) bonds cleaved Base –3 N NaOH, 6 hr at 100 C° All peptide (and amide) bonds cleaved, but hydroxyl groups and guanidino groups are destroyed Enzymes –proteolysis; incomplete due to specificity of enzymes Use hydrolysis to answer 2 questions: 1) determine amino acid composition 2) Help determine entire amino-acid sequence by fragmenting into small peptides Primary structure * Fred Sanger 1918-2013 1958 Nobel Prize 110.9 107.8 110.5 * Dalton (D) = amu = g/mol kD = kilodalton = 1000 D 1 residue (aa) ~ 110 D (0.11 kD) Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends 5) Divide into fragments and determine sequence 6) Divide into different set of fragments and determine sequence 7) Determine overlaps and piece original sequence back together 27 Ion Exchange Chromatography: Example of aminoacid mixture Basis = charge cation negatively If this were a mixture of G, K, and D, what would the order of elution be? 1st= D, 2nd = , 3rd = K Determination of primary structure Amino Acid Composition Absorbance is at 570 nm (purple). How did they make the amino acid residues colored? …......ninhydrin How many AA are “resolved?” Where are the other 3? …....17 …....Asn & Gln have already been converted to Asp & Glu, respectively ........enough Trp was not in this protein to ”see“ Determination of primary structure Amino Acid Composition Cation exchanger=Dowex-50 (sulfonic acid on polystyrene) Calculate the “mole%” or “mole fraction”; in other words the stoichiometry: Example; protein MW = 11,000 Da Approximate #AA = 11,000/110 (Ave MW for AA) = 100 AA If 10% of A570 is 10% for Arg: 100 x 0.10 = 10 Arg in protein Determination of primary structure 1) Purify protein 2) Determine the amino-acid composition, including stoichiometry 3) Disrupt structure (2°, 3°, 4°, and disulfides) 4) Determine the number of peptide chains by counting number of amino terminal ends 5) Divide into fragments and determine sequence 6) Divide into different set of fragments and determine sequence 7) Determine overlaps and piece original sequence back together 33